RNA TUMOR VIRUS--DNA INTEGRATION/GENE EXPRESSION CONTROL

拟议研究的长期目标是了解 RNA肿瘤病毒控制自身表达的机制 并影响宿主细胞中基因的表达。 具体目标包括研究逆转录病毒DNA的生物化学。 病毒基因组序列的整合、鉴定和分析 控制多聚蛋白和RNA的加工。禽肉瘤/白血病病毒 (ASLV)作为主要的模型系统。然而，比较研究 与人类免疫缺陷病毒(艾滋病毒)的合作也在计划之中。 对于整合的研究，实验策略包括分析 病毒颗粒分离蛋白的相关生化活性 并通过重组DNA技术在异源系统中生产。 将利用异源系统的能力来提供 大量成熟的活性蛋白质和功能基因片段 会受到定点突变的影响。多种DNA底物 包含假定的病毒整合位点将在体内进行测试，使用 敏感的基因检测以检测重组事件并评估 在体外系统中进行研究，其中包括细胞提取物和病毒- 感染和/或纯化的蛋白质和核成分。这种蛋白质 处理研究将受益于我们不断增长的对 逆转录病毒蛋白水解酶的生化和三维结构。 突变体将基于这一知识进行设计，并将用于 评估蛋白水解酶活性对病毒核心的贡献 形态发生。遗传和生化方法也将用于 鉴定和鉴定参与控制的病毒RNA序列 并确定这种调控的机制。为了这些 研究，强大的基因选择成为可能，因为有了 独特的病毒突变体将提供特殊的优势。 这项工作的结果将有助于理解病毒和 逆转录病毒诱发癌症的细胞生化反应。在……里面 此外，逆转录病毒是一种很有希望扩大的工具。 我们对其他情况下涉及的一般机制的理解 基因损伤、基因扩增或基因异常的致癌作用 重排和控制可能会引发恶性反应。

The long term objective of the proposed research is to understand the mechanism by which RNA tumor viruses control the expression of their own genomes and influence the expression of genes in their host cells. Specific aims include studies of the biochemistry of retroviral DNA integration and identification and analysis of viral genomic sequences that control polyprotein and RNA processing. The avian sarcoma/leukosis viruses (ASLV) serve as the primary model systems. However, comparative studies with human immunodeficiency virus (HIV) are also planned. For studies of integration, experimental strategies include analysis of the relevant biochemical activities of proteins isolated from viral particles and produced in heterologous systems through recombinant DNA techniques. The heterologous systems will be exploited for their ability to provide large amounts of mature, active proteins and functional gene fragments that can be subjected to site-directed mutagenesis. A variety of DNA substrates containing putative viral integration sites will be tested in vivo using sensitive genetic assays to detect recombination events and to evaluate the investigated in vitro systems, which include cell extracts and virus- infected and/or purified protein and nucleic components. The protein processing studies will benefit from our growing knowledge of the biochemistry and three-dimensional structure of the retroviral protease. Mutants will be designed based on this knowledge and will be used to evaluate the contribution of protease activity to virion core morphogenesis. Genetic and biochemical approaches will also be used to identify and characterize viral RNA sequences involved in the control of RNA processing and to determine the mechanism of this control. For these studies, powerful genetic selection made possible by the availability of unique viral mutants will provide a special advantage. Results from this work will contribute to an understanding of the viral and cellular biochemical reactions that underlie retrovirus-induced cancer. In addition, the retroviruses represent a promising tool with which to enlarge our understanding of the general mechanisms involved in other instances of oncogenesis where genetic damage, gene amplifications or abnormal gene rearrangements and controls can trigger malignancy.